Ice formation is damaging to living systems and food products and may be a nuisance and a hazard to human beings who must cope with snow and ice in their environment. The field of the present invention is the provision of processes for the preparation of specific chemical agents, referred to herein as ice interface dopants (IID), that will effectively reduce ice formation and make ice that does form innocuous to living systems and foodstuffs and less troublesome and hazardous to humans and machinery in the environment.
Referring to FIGS. 1A-B, ice crystallizes in the shape of a hexagonal plate 10. A plane defined by the a axis 12 and the b axis 14 (which is crystallographically identical to the a axis) and perpendicular to the c axis 16 defines a hexagonal cross section called the basal plane 18. The six faces of the hexagon are called prism faces 20. Crystallographically, the basal plane 18 is referred to as the 0001 surface, and the prism face is referred to as the 1100 surface or the 1120 surface depending on the orientation.
FIGS. 2A-D show that the units of the crystal that give rise to this macroscopic structure are also hexagonal. In FIGS. 2A-D show, following common usage, only the oxygen atoms are represented. The hydrogen atoms lie along the straight lines shown bonding each oxygen atom to its four nearest neighbors.
FIG. 2A shows the basal plane 0001 surface as seen from above. Within each hexagon, three vertices project upward (or forward), and the three intervening vertices project downward (or backward). The upward vertices are separated by 4.5 .ANG..+-.0.02 .ANG. and are located at a 60.degree. angle with respect to each other. Their fourth bonds extend perpendicularly out of the page toward the viewer. Another spacing at 7.36 .ANG. separates alternate bilayers 21 of oxygen atoms in the lattice, or, viewed differently, separates each oxygen-defined hexagon from an identical hexagon located immediately adjacent to it. FIG. 2B shows views of the crystallographic 1100 and 1120 prism faces.
Several natural molecules exist that alter the behavior of ice and of water. Antifreeze glycoproteins (AFGPs) and antifreeze proteins or antifreeze peptides (AFPs) produced by several species of fish are believed to adsorb preferentially to the prism face 20 of ice and thus to inhibit ice crystal growth perpendicular to the prism face, i.e, in the direction extending along the basal plane 18 and along the a and b axes 12 and 14.
This capability is sufficient to permit certain fish to live their entire lives at a body temperature about 1.degree. C. below the thermodynamic freezing point of the fishes' body fluids. These fish can ingest and contact ice crystals that might otherwise provide crystal nucleation sites without being invaded by the growth of ice through their supercooled tissues because the AFGPs present in their tissues and body fluids block ice growth despite the presence of supercooling. Insect antifreeze or "thermal hysteresis" proteins (THPs) are even more effective, being active at supercooling levels of 2.degree. C. or more below the thermodynamic freezing point.
The natural "antifreeze" or "thermal hysteresis" proteins found in polar fish and certain terrestrial insects are believed to adsorb to ice by lattice matching (Davies and Hew, FASEB J., 4; 2460-2468, 1990) or by dipolar interactions along certain axes (Yang, Sax, Chakrabartty and Hew, Nature, 333:232-237, 1988).
Antifreeze glycoproteins (AFGPs) and antifreeze proteins or antifreeze peptides (AFPs) found in certain organisms provide natural "proofs of principle" for the concept of novel man-made IIDs. However, natural ice interface doping proteins are not sufficiently active or abundant for most practical applications of interest. Furthermore, a disadvantage of basal plane growth inhibition is that, when supercooling becomes sufficient to overcome ice crystal growth inhibition, growth occurs, by default, predominantly in the direction of the c axis 16, perpendicular to the basal plane 18. This results in the formation of spindle or needle-shaped ice crystals (FIG. 1B) that are more damaging to living cells than normal ice, apparently for mechanical reasons.
Natural IIDs are commercially available only in a very limited quantity and variety. Furthermore, they must have fairly high relative molecular masses (typically at least about 5,000 daltons) to be effective. This tends to make them expensive, and they often require complex interactions with other hard-to-acquire proteins and often require carbohydrate moieties for full effectiveness.
Furthermore, addition of natural fish AFGP to a concentrated solution of cryoprotectant (30-40% v/v DMSO) had minimal effect on ice crystal growth rates below -20 to -40.degree. C. (Fahy, G. M., in Biological Ice Nucleation and its Applications, chapter, 18, pp. 315-336, 1985), thus making questionable its effectiveness for use in organ vitrification for cryopreservation.
Another problem with natural antifreeze proteins is that continuing confusion over their precise mechanisms of action hampers the development of recombinant variants that could be more effective. Recently, Warren and colleagues reported some progress in this direction (U.S. Pat. No. 5,118,792).
The concept of designing specific artificial chemical agents whose purpose is to control the physics of ice was first mentioned by Fahy in Low Temperature Biotechnology, McGrath and Diller, eds., ASME, pp.113-146, 1988. The sole mention of this idea was the single statement that "insight into the mechanism of AFP action . . . opens the possibility of designing molecules which may be able to inhibit ice crystal growth in complementary ways, e.g., along different crystallographic planes." However, no method of preparing such molecules was suggested.
Kuo-Chen Chou ("Energy-optimized structure of antifreeze protein and its binding mechanism", J. Mol. Biol., 223:509-517, 1992) mentions an intention to specifically design ice crystal growth inhibitors. However, it is confined to minor modifications of existing antifreeze molecules, and does not envision the present radically different approach of preparing synthetic IIDs de novo.
Based on these observations, it is advantageous to design molecules that can prevent ice crystal growth specifically in the direction of the c axis in accordance with the present invention. When used in combination with an agent acting to block growth in the direction of the basal plane, such that all growth planes would be inhibited rather than only one, such an agent should avoid the lethal drawbacks of the prior art of freezing cells using only basal plane growth inhibitors. Furthermore, since growth in the direction of the c axis, hereinafter "C growth," is the limiting factor for supercooling in the presence of agents that adsorb to the prism face (agents that block growth in the a axis direction, or "A growth"), C growth inhibitors should enhance supercooling considerably over the supercooling achievable with A growth inhibitors alone when used in combination with A growth inhibitors. For this reason, although the principles described herein permit IIDs to be designed to bind to any crystallographic plane of ice desired whatever, or even to non-crystallographic patterns inherent in the ice crystal structure, the specific molecular prototypes described herein have been designed specifically to bind to the basal plane so as to prevent C growth.
A problem with natural antifreeze proteins has been continuing confusion over their precise mechanisms of action. Recently, Sicheri and Yang (Nature 375: 427-431, 1995) described a clear model of how AFPs undergo lattice matching with ice. They indicated that, of 8 AFPs examined, the number of ice-binding atoms ranged from 3 to 10 per AFP and that each AFP formed, on average, ice contacts at between 1 in every 4.8 to 1 in every 15 amino acids present in the molecule (roughly 1 ice bond per 422-1340 daltons of AFP mass). The ice-binding amino acids were threonine, aspartate (asp), asparagine (asn), and lysine. Each binding amino acid formed one bond per amino acid and the bonds were formed by the hydroxyl oxygen of threonine, the amino nitrogen of lysine and of asparagine, and the acid oxygen (O.sup.- or carbonyl 0) of aspartate. For the winter flounder AFP, detailed analysis showed that the lattice matching depended on a planar arrangement of the AFP's bonding groups and on geometrical constraints on the freedom of motion of the matching groups. Bonding took place on the ridges of the 2021 plane (Biophys. J. 63: 1659-1662, 1992; Faraday Discuss. 95: 299-306, 1993; J. Am. Chem. Soc. 116: 417-418, 1994.) More detailed analysis showed that the lattice match between asn and asp oxygen and nitrogen and ice oxygens was imperfect. For one thing, the oxygens in ice associated with these sites were located to the side of each binding atom, not directly underneath. For another, the trigonal planar (sp2) coordination of the hydrogen-bonding groups of asn and asp differ from the tetrahedral (sp3) coordination of oxygens in ice. They concluded that "the underlying hydrogen-bonding interactions are likely to be more liberally defined than previously proposed" by other authors (Biophys. J. 59: 409-418, 1991; Biophys. J. 63: 1659-1662, 1992; Biophys. J. 64: 252-259, 1993).